The present invention, in some embodiments thereof, relates to pore-forming polypeptides that are naturally plugged and are engineered to open by the action of proteases. The plugged pore-forming polypeptides may be incorporated at the surface of encapsulating particles such that they generate pores in the particles only upon exposure to specific proteases. Opening of the pores releases the content of the encapsulating particle.
Cancer is currently the second leading cause of death in the United States. 85% of cancer patients have solid tumors and 50% of those patients die as a result of malignant disease. Local control of the tumor is particularly difficult in the cervix, colon, ovarian, pancreas, and brain. There is hence an urgent and currently unmet need to improve eradication of primary solid tumors and solid metastases.
Most chemotherapeutic drugs act on both normal as well as cancerous tissues. As such, one of the challenges in treating cancerous tumors with chemotherapy is maximizing the killing of cancer cells while minimizing the harming of healthy tissue. Depending on the administration route (e.g., intravenous) and nature of the drug (e.g., its physical and pharmacokinetic properties), oftentimes only a small fraction of the dose reaches the target cells; the remaining amount of drug acts on other tissues or is rapidly eliminated.
Standard chemotherapeutic drugs (e.g., Doxorubicin, Camptothecin, Paclitaxel, and Palatinate) are usually injected systemically to the patient and act by preventing the proliferation of the cancer cells. These drugs must be administered at low doses since they are very aggressive and have many harsh side effects. Doxorubicin, for example, has limited specificity, killing all fast replicating cells, by: intercalating into the DNA to prevent its unwinding by Topoisomerase II and inhibiting macromolecule synthesis. As such, many healthy cells including white blood cells, gastrointestinal cells, and hair follicles are also susceptible to the effects of the drug, leading to pathophysiological conditions such as neutropenia, stomatitis, and alopecia. Some new drug treatments utilize chemoprotective agents to help prevent side effects by protecting healthy cells against the drug's toxicity. Other treatments reduce side effects by improving the chemistry of the drug, while some improve the drug-delivery to the cancer cells.
To improve delivery efficiency and reduce toxicity to non-target cells, various strategies have been used to deliver drugs to specific sites in the human body. Tumor cells are often characterized by a specific expression pattern of membrane associated proteins such as receptors, membrane transport systems or adhesion molecules. Provided that these structures are accessible from the extracellular milieu, such properties can be exploited for an active targeting of diseased cells and tissues using specific effector molecules. The concept of active targeting has the potential to combine the advantage of an increased therapeutic efficacy with a reduced risk for adverse side-effects in non-diseased tissues. With the arrival of genetic engineering technologies, which made it possible to design chimeric mouse-human monoclonal antibodies or recombinant peptidic receptor ligands, the clinical use of these active tumor targeting strategies has become reality.
Various carriers have been explored including liposomal vectors, micelles, carbon nanotubes, polymeric nanoparticles, polymer conjugates, hyalronan-shelled bodies, and lipidic cubic phases for increasing the delivery of a chemotherapeutic drug to a target site.
The use of liposomes is particularly advantageous since its pharmacokinetic properties can be modulated by specific modifications of the liposome surface. Besides direct chemical modifications of the phospholipid headgroups (such as the introduction of surface charges or hydrophilic groups, conjugation of proteins, peptides or other macromolecules to the liposome surface can be achieved. Chemical conjugation techniques provide thereby a stable link between the liposomal phospholipids and a specific targeting vector. The availability of pegylated liposomes made the development of vector-conjugated liposomes possible since the unique properties of these long-circulating liposomes can be combined with those of a targeting vector of choice within one preparation.
Triggered release of drugs and labeled molecules from liposomes has been recognized to be an attractive therapeutic approach. In this approach of drug delivery, the drug delivery vehicles do not release contents until the vehicle's membranes are destabilized by the external agents (trigger). The trigger can be a change in mechanical stress, metal ions, or enzymes such as elastase, alkaline phosphatase, trypsin and phospholipase A2. Conformational changes of peptides, induced by the change in pH, have also been used to facilitate the content release from liposomes.
U.S. Patent Application 20060210549 teaches triple helix trigger peptides which destabilize a liposome preparation on contact with a protease enzyme. These helical peptides do not create pores per se and further are devoid of plugging modules.
U.S. Patent Application 20090016988 teaches protease activated pore forming toxins as pharmaceutical agents for the treatment of cancer. The toxins are multisubunit proteins and do not comprise a naturally occurring plugging module.
U.S. Patent Application 20040180094 teaches protease-activated pore forming toxins as triggers for the release of agents inside an encapsulating vehicle. The toxins are multisubunit proteins and do not comprise a naturally occurring plugging module.
Richard Lipkin [Science News, Cell Membrane Research Sep. 24, 1994] summarizes the review of Hagan Bayley [Bioorganic Chem 23, 340-354 (1995)] who teaches selective release of a drug from an encapsulating vehicle using a trigger which comprises a protease-activated multisubunit pore-forming toxin (alpha hemolysin). Bayley does not teach the use of pore-forming proteins which are not toxins and comprise naturally occurring plugging modules.